Effects of triclosan on the early life stages and reproduction of medaka Oryzias latipes and induction of hepatic vitellogenin

Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) is widely used as antibacterial agent in various industrial products, such as textile goods, soap, shampoo, liquid toothpaste and cosmetics, and often detected in wastewater effluent. In this study, the effects of TCS on the early life stages and reproduction of medaka (Oryzias latipes) were investigated. The 96-h median lethal concentration value of TCS for 24-h-old larvae was 602 microg/l. The hatchability and time to hatching in fertilized eggs exposed to 313 microg/l TCS for 14 days were significantly decreased and delayed, respectively. An assessment of the effects of a TCS 21-day exposure period on the reproduction of paired medaka showed no significant differences in the number of eggs produced and fertility among the control and 20, 100 and 200 microg/l TCS treatment groups. However, concentrations of hepatic vitellogenin were increased significantly in males treated with TCS at 20 and 100 microg/l. In the F(1) generations, although the hatching of embryos in the 20 microg/l treatment showed adverse effects, there was no dose-response relationship between hatchability and TCS treatment levels. These results suggest that TCS has high toxicity on the early life stages of medaka, and that the metabolite of TCS may be a weak estrogenic compound with the potential to induce vitellogenin in male medaka but with no adverse effect on reproductive success and offspring.     

How estrogenic is nonylphenol? A transgenerational study using rainbow trout (Oncorhynchus mykiss) as a test organism

The aim of the present study was to evaluate both estrogenic effects in directly NP-exposed sexually mature rainbow trout and possible transgenerational effects in the offspring of exposed fish. Four months prior to spawning, adult rainbow trout of both sexes were exposed intermittently to NP concentrations of 1 and 10 microg/l. At the end of the exposure period, which coincided with the beginning of spawning time, vitellogenin levels in the plasma of adult male rainbow showed a significant increase compared to the control group. After exposure to 10 microg NP/l reproduction was impaired as indicated by significantly reduced hatching rates. Histological examination of the testicular tissue of NP-exposed individuals revealed no morphological differences from the controls. In the offspring, vitellogenin levels of male individuals were not affected, whereas in females they were significantly higher than in the control progeny. The histological examination revealed no alteration in sex ratios. In single cases, intersex occurred in both male and female offspring of exposed fish. The analysis of sex steroid levels revealed a two-fold increase of estradiol in the plasma of male offspring and a 13-fold elevation of testosterone in the plasma of female progeny. The present findings indicate that NP, in an environmentally relevant concentration range, acts as a weak estrogen in directly exposed adult male rainbow trout as indicated by elevated plasma vitellogenin levels. Reproduction success was reduced as indicated by decreased hatching rates. Hormonal imbalances detected in the offspring of exposed fish indicate a transgenerational effect mediated by the endocrine system.     

Diethylstilbestrol at environmental levels affects the development of early life stage and target gene expression in Japanese Medaka (<Emphasis Type="Italic">Oryzias latipes</Emphasis>)

In this study, the biologic effects of DES on the early life and adult life stages of Japanese medaka (Oryzias latipes) were evaluated. At the early life stage, the fertilized eggs were exposed to 1–1000 ng/L diethylstilbestrol (DES) for 15 days and the hatched larvae were continually exposed to the same concentrations for an additional 25 days. Significant adverse effects on hatchability, time to hatching and mortality rate occurred at DES concentrations of 100 and 1000 ng/L, while the abnormality (scoliosis and abdominal swelling) rate was significantly increased at 10 ng/L and above. After exposure, the fish were maintained in charcoal-dechlorinated tap water for a further 30 days. Only the male gonadosomatic index (GSI) at 1000 ng/L was significantly increased. At concentrations greater than 1 ng/L, estrogen receptor α (ERα) mRNA in both sexes and vitellogenin-I (Vtg-I) mRNA in males were significantly down-regulated; while Vtg-I mRNA in females was significantly up-regulated. When sexually mature medaka were exposed to 10 and 1000 ng/L DES for 21 days, only the GSI in females was significantly decreased at 1000 ng/L. At 10 and 1000 ng/L, ERα mRNA in both sexes was significantly down-regulated, while Vtg-I mRNA in males was significantly up-regulated. These findings showed that DES at the environmental concentration of 10 ng/L can affect the early life stage development of medaka and alter liver ERα and Vtg-I gene expression. Therefore, if we only focused on these sensitive toxicity endpoints such as ERα and Vtg-I mRNA expression, DES has a strong estrogenic effect on Japanese medaka.     

Tamoxifen effects on the early life stages and reproduction of Japanese medaka (Oryzias latipes)

Tamoxifen is widely used in the treatment of breast cancer and can enter the aquatic environment in municipal wastewater. To evaluate potential effects on embryonic development of Japanese medaka, fertilized eggs were exposed to tamoxifen at 1-625μg/l for 14 days. Adverse effects on hatchability and time to hatching only occurred at 125 and 625μg/l. Reproductive effects were assessed by exposing adults for 21 days to the same dose range. At all concentrations tested, tamoxifen significantly increased plasma vitellogenin levels in males in a dose-dependent manner. Fecundity and fertility were detrimentally affected at 625μg/l. Additionally, F1 eggs were removed from tamoxifen-contaminated water to evaluate transgenerational effects. Hatchability was affected at 625μg/l but no morphological deformities were observed. A significant dose-dependent increase in the proportion of genotypic males occurred at all concentrations greater than 5μg/l.     

Estrogenicity of butylparaben in rainbow trout Oncorhynchus mykiss exposed via food and water

The estrogenic effect of butylparaben was investigated in a rainbow trout Oncorhynchus mykiss test system. Butylparaben was administered orally to sexually immature rainbow trout every second day for up to 10 days in doses between 4 and 74 mgkg(-1)2d(-1) and in the water at 35 and 201 microgl(-1) for 12 days. Plasma vitellogenin was measured before and during the exposures and the concentrations of butylparaben in liver and muscle were determined at the end of experiments. Increases in average plasma vitellogenin levels were seen at oral exposure to 9 mg butylparaben kg(-1)2d(-1). The ED50 values for increase in vitellogenin synthesis were 46, 29 and 10.5 mg butylparaben kg(-1)2d(-1), respectively, at day 3, 6 and 12. Exposure to 201 microg butylparaben l(-1) increased vitellogenin synthesis, but exposure to 35 microgl(-1) did not. Butylparaben showed little tendency to bioaccumulation in rainbow trout; less than 1 per thousand of the total amount of butylparaben administered orally at 51 mgkg(-1)2d(-1) over the 12 days experimental period was retained in liver at the end of the experiment. After 12 days exposure to 35 and 201 microg butylparaben l(-1), plasma concentrations were 9 and 183 microgl(-1), respectively, and for the fish exposed to 201 microgl(-1) there was a positive correlation between concentrations of vitellogenin and butylparaben in the plasma. On the assumption that butylparaben removed from the water phase during water exposure were taken up into the fish, butylparaben uptake rates in the fish exposed to 35 and 201 microg butylparaben l(-1) were 13 and 78 mgkg(-1)day(-1), respectively.     

Effect of aluminium and calcium ions on survival and physiology of rainbow trout Salmo gairdneri (Richardson) eggs and larvae exposed to acid stress

Survival and different physiological parameters of developing rainbow trout were investigated during exposure to conditions of low pH, different calcium and aluminium ion levels. In fertilized non-hatched eggs from rainbow trout exposed to different low pH levels mortality occurred at regular intervals during development. Hatching rate was delayed at the lowest ambient pH levels and hatching success was reduced accordingly. The lethal concentration of aluminium ions (LC₅₀) was 3.8 mg Al/l in soft water and 71 mg Al/l in hard water, when determined on day 25 after hatching at pH 7. In general, exposure to aluminium reduced survival. However, the greatest reduction in survival was observed in relation to the calcium ion concentration of the ambient medium. Exposure to Al reduced dry weight by 9% and respiration rate by 13% in soft water at pH 5. Aluminium ions had only minor effects on the body concentration of Ca and on the cardiac rate. Respiration rate was reduced by 41% by a lowering of pH only, and low pH also had the greatest reducing effect on cardiac rate (32% at pH 5 in soft water). Previous exposure to low pH, low calcium and high aluminium ion concentrations had no significant effect on larval growth after transfer to hard water at pH 7.     

Characterization of responses to the antiandrogen flutamide in a short-term reproduction assay with the fathead minnow

A short-term reproduction assay with the fathead minnow (Pimephales promelas) has been developed to detect chemicals with the potential to disrupt reproductive endocrine function controlled by estrogen- and androgen-mediated pathways. The objective of this study was to use the assay to characterize responses of fathead minnow reproductive endocrinology and physiology to the mammalian antiandrogen, flutamide. Male and female fish were exposed to nominal (target) concentrations of 50 and 500 microg flutamide/l for 21-days, following which plasma steroid and vitellogenin concentrations were determined and gonadal morphology assessed. Fecundity of the fish was significantly reduced by exposure to a measured test concentration of 651 microg flutamide/l. In addition, embryo hatch was significantly reduced at this concentration. Qualitative histological assessment of ovaries from females exposed to flutamide indicated a decrease in mature oocytes and an increase in atretic follicles. Testes of males exposed to flutamide exhibited spermatocyte degeneration and necrosis. Concentration-dependent increases in plasma testosterone and vitellogenin concentrations were observed in the females. Flutamide also altered reproductive endocrinology of male fathead minnows. Males exposed to 651 microg flutamide/l exhibited elevated concentrations of beta-estradiol and vitellogenin. In summary, the results of this study with the fathead minnow demonstrate that flutamide affects reproductive endocrine function in fish and that the type of hormonal pattern and histopathology effects observed are consistent with an antiandrogenic mode-of-action. Consequently, our findings suggest that the 21-day reproduction assay utilizing fathead minnows is a sensitive short-term screening method for the detection of endocrine-disrupting chemicals, including antiandrogens.     

Bisphenol A causes mortality and reduced hatching success through increase of cell damage and dysfunction of antioxidant defense system in marine medaka embryo

In this study, marine medaka embryos were continuously exposed to different concentrations of bisphenol A (BPA; 200-1000 μg/L) during post fertilization. Mortality, hatching rate, and cellular death in marine medaka embryos were detected. In addition, survival rate of hatched larvae was measured to analyze whether BPA has persistent effects from BPA-exposed embryos to post-hatching period. Our results clearly showed that the hatching rate was decreased in a dose-dependent manner, while the mortality was increased with induction of cellular death and dysfunction of antioxidant defense system in marine medaka embryos. Of hatched larvae, the survival rate was decreased as the exposure concentrations increasing. Thus, our data suggested that early exposure of BPA caused embryonic toxicity, resulted in persistent effects on larval survival in marine medaka.     

The effect of 4-nonylphenol on semen quality, viability of gametes, fertilization success, and embryo and larvae survival in rainbow trout (Oncorhynchus mykiss)

The present study investigated in vivo and in vitro effects of environmental relevant concentrations of 4-nonylphenol (100-750 ng l(-1)) on the reproduction of rainbow trout (Oncorhynchus mykiss). To determine the effect of 4-nonylphenol on semen quality rainbow trout were exposed to three concentrations of 4-nonylphenol in a flow-through system during the spawning period (60 days). At an estimated 4-nonylphenol concentration of 750 ng l(-1) semen production was completely inhibited, at 280 and 130 ng l(-1) the semen production was significantly reduced in comparison to the control. Sperm density, sperm motility and sperm fertility were not affected. Also the development of embryos and larvae at the end of yolk sac stage was affected by 4-nonylphenol. At estimated 4-nonylphenol exposure levels of 280 and 750 ng l(-1) the percentage of eyed stage embryos was slightly but significantly lower (2-4%) than at 130 ng l(-1) 4-nonylphenol and in the control. At 4-nonylphenol concentrations of 750 ng l(-1) only 23.8 +/- 1.2% of the larvae survived to the end of the yolk sac stage, at 280 ng l(-1) 53.7 +/- 8.2%, at 130 ng l(-1) 73.8 +/- 1.5%, and in the control 70.9 +/- 1.8%. Sperm motility was not affected by 4-nonylphenol as sperm motility rate, swimming velocity, swimming pattern and motility duration were similar in water and in water containing of 100, 250, or 750 ng l(-1) 4-nonylphenol. Incubation of eggs in physiological saline solution containing of 100, 250, or 750 ng l(-1) 4-nonylphenol did not change their fertilizability in comparison to the control. Therefore, 4-nonylphenol did not affect the egg viability. Also the fertilization process (sperm egg contact) was not influenced by 4-nonylphenol as the fertilization rate (percentage of hatched larvae) was similar to the control when eggs were fertilized in water containing of 100, 250, or 750 ng l(-1) 4-nonylphenol.     

Tissue-specific Cu bioaccumulation patterns and differences in sensitivity to waterborne Cu in three freshwater fish: rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio), and gibel carp (Carassius auratus gibelio)

Rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio) were exposed to copper (1-20 microM) in softened Antwerp City tap water at pH 7.3 +/- 0.1 and with a water hardness of 292.4 +/- 8.1 mg/L CaCO(3) (Ca 100.8 +/- 3.0 mg/L; Mg 11.0 +/- 0.2 mg/L). LC50s (96 h) were determined and copper accumulation in gills, liver, and kidney assessed over a 10-day period. Rainbow trout (96 h LC50: 3.3 microM/210 microg/L) were three times more sensitive to Cu exposure than common carp (96 h LC50: 10.4 microM/661 microg/L) and almost seven times more sensitive than gibel carp (96 h LC50: 22.0 microM/1398 microg/L). After 96 h, the incipient lethal level (ILL) was reached for common carp, and by the end of the experiment (>120 h) also for rainbow trout. The ILL was never reached for gibel carp. Survival analysis confirmed the differences in sensitivity shown by the 96 h LC50 values. At 1 microM Cu, the relative risk to die was six to seven times greater for rainbow trout as for common or gibel carp, respectively, while it was 9000 and 19,000 times greater at 5 microM Cu. Only the environmental Cu concentrations contributed significantly (P < 0.001) to the Time-To-Death (TTD). Tissue Cu concentrations did not relate to TTD. Among species, a clear difference in metal handling was apparent, with high liver residues and liver accumulation rates for the most sensitive species, the rainbow trout, and lower liver but higher kidney residues and kidney accumulation rates for the most resistant species, the gibel carp. Gill concentrations and accumulation rates were lowest in the sensitive rainbow trout.     

Evaluation of estrogenic activities of aquatic herbicides and surfactants using an rainbow trout vitellogenin assay.

Estrogenic potencies of four herbicides (triclopyr, 2,4-dichlorophenoxyacetic acid (2,4-D), diquat dibromide, glyphosate), two alkylphenol ethoxylate-containing surfactants (R-11 and Target Prospreader Activator (TPA)), and the binary mixture of surfactants with the herbicides were evaluated using an in vivo rainbow trout vitellogenin assay. Juvenile rainbow trout exposed to 2,4-D (1.64 mg/l) for 7 days had a 93-fold increase in plasma vitellogenin (Vtg) levels compared with untreated fish, while rainbow trout exposed to other pesticides alone did not show elevated vitellogenin levels compared to the control fish. When combined with surfactants, trends indicated enhanced estrogenicity for all combinations, but only 2,4-D and triclopyr caused significant induction of Vtg. Concentration-response studies demonstrated that the lowest observed effect concentrations (LOECs) for 2,4-D and triclopyr were 0.164 mg/l and 1 mg/l, respectively. In terms of measured 4-nonylphenol (4-NP), the LOECs of R-11 and TPA were 20 micro/l and 9.5 microg/l, respectively. Binary mixtures of TPA and 2,4-D showed a greater than additive estrogenic response at the lowest concentrations tested, but a less than additive response at the highest combined concentrations. Binary mixtures of TPA with triclopyr also caused greater than additive Vtg responses in two middle concentrations when compared to TPA or triclopyr alone. When trout were exposed to water collected from a site where triclopyr was used in combination with TPA, a concentration-dependent increase in Vtg expression was observed. Measured values of 4-NP were 3.7 microg/l, and triclopyr concentrations were below detection (<5 ng/l). Estradiol equivalents (EEQs) of the lake water were calculated from an estradiol concentration-response curve and were similar (8.5 +/- 7.7 ng/l) to the mean values for the combined triclopyr + TPA treatments (9.9-12.2 ng/l) in the laboratory, suggesting the estrogenicity of the water may have been due to the treatment. These results demonstrated the binary mixture of alkylphenol ethoxylate-containing surfactants with two aquatic pesticides possessed greater than additive estrogenic responses in fish under laboratory conditions and in a field setting.     

The effects of methyltestosterone on the sexual development and reproduction of adult medaka (Oryzias latipes)

We examined the effects of methyltestosterone (MT) on the reproduction of medaka (Oryzias latipes). Six mating pairs of medaka were exposed for 3 weeks to MT at the measured concentrations of 22.5, 46.8, 88.1, 188, and 380 ng/L. The fecundity and fertility of paired medaka were significantly decreased in the 46.8, 88.1, 188, and 380 ng/L MT groups compared with those of controls. The gonadosomatic indices of female fish exposed to >or=46.8 ng/L were increased significantly. Gonadal histology showed degeneration of oocytes in the ovaries of fish in all MT treatment groups. Hepatic vitellogenin concentrations were significantly decreased in female fish treated with MT at 188 and 380 ng/L, and the hatchability and survival rate of the offspring were decreased in all MT treatment groups. These results clearly demonstrate that MT at >or=46.8 ng/L inhibited gonadal development and adversely affected the reproduction of medaka.     

The Chinese rare minnow (Gobiocypris rarus) as an in vivo model for endocrine disruption in freshwater teleosts: a full life-cycle test with diethylstilbestrol

Chinese rare minnow (Gobiocypris rarus), a freshwater teleost, was exposed to diethylstilbestrol (DES) at 0.05, 0.5, 1 and 5 microg/L from fertilized eggs for up to mature period under flow-through condition. Several endpoints that related to development, reproductive fitness and transgenerational effects were evaluated. It was found that body length and body weight were significantly reduced and vitellogenin (Vtg) levels were significantly increased for fish exposed to DES. Histological examination showed that the sex ratios of F0 fish skewed to female and about 2% of the fish exposed to 0.05 microg/L DES developed testes-ova. The reproductive success, as determined from data on egg production, was reduced in female fish exposed to 0.05, 0.5, 1 and 5 microg/L DES. The lowest-observed-effect concentrations (LOEC) for changes of sex ratios, reproductive success and histology alteration of F0 are 0.05 microg/L. In the offspring, transgenerational effects on egg hatching rate, egg fertilization and Vtg levels of juvenile individuals were not observed. However, survival of F1 generation fry significantly declined. The analysis of sex steroid levels revealed a significant decrease of testosterone (T) in the whole body homogenates (WBH) of male progeny and somewhat elevation of estradiol (E2) in the WBH of female offspring. These findings indicate that exposure to DES causes a variety of developmental, reproductive and transgenerational effects.     

Uptake, metabolism and excretion of bisphenol A in the rainbow trout (Oncorhynchus mykiss)

The uptake, metabolism and excretion of the oestrogenic chemical bisphenol A (BPA) were studied in juvenile rainbow trout (Oncorhynchus mykiss). BPA was detectable in plasma, liver and muscle after 2 h of water exposure at 0.44 microM (100 microg BPA/l), and a steady state was reached within 12-24 h. The concentration of the glucuronidated degradation product in the plasma was about twice that of the parent compound. A plasma half life of BPA was calculated as 3.75 h following injection of the compound. The vitellogenin synthesis was measured in response to the BPA treatment, and a lag period of 5 and 7 days between injection of the compound and a significant vitellogenin response was observed for females and males, respectively. At the time of the vitellogenin response no BPA could be detected in the liver tissue from either male or female fish. These results indicate that fish briefly exposed to elevated levels of oestrogenic chemicals might develop a response several days later.     

Responses of Daphnia magna to pulsed exposures of arsenic

Research on the toxicity of arsenic has focused on sublethal effects that do not provide sufficient information for risk estimation. While most studies have focused on organism response to constant arsenic exposures, organisms in nature are exposed to fluctuating As concentrations. Consequently, results obtained from standardized bioassays with constant exposures may not adequately characterize risk to indigenous biota. This research was designed to characterize the response of Daphnia magna to fluctuating arsenic exposures during 21-day experiments. At concentrations > or =3000 microg/L As, 21-day pulsed exposure mortality increased as a function of exposure concentration and duration. In addition, 21-day pulsed exposure mortality increased with increasing recovery time. Pulsed As exposure did not affect the growth of D. magna over 21 days. Twenty-one day accumulative reproduction of D. magna was only affected by pulsed exposures of high As concentration and long durations.     

Effects of long-term nonylphenol exposure on gonadal development and biomarkers of estrogenicity in juvenile rainbow trout Oncorhynchus mykiss

Environmental pollutants with estrogenic activity including nonylphenol (NP) have the potential to alter gonadal development and reproduction of wild fish. To investigate the estrogenic action of environmentally relevant concentrations of NP, rainbow trout (Oncorhynchus mykiss) were continuously exposed during the embryonic, larval and juvenile life stage to 1.05 and 10.17 μg/l NP for 1 year, and sexual differentiation, vitellogenin (VG), VG mRNA, and zona radiata protein (ZRP) expression were examined after that period. The applied NP concentrations did not affect mortality and hatching rates, and did not have an influence on the body weight of 1-year-old fish. No occurrence of testis–ova was observed and sex-ratios of NP exposed groups of fish were unchanged when compared with control groups. The induction of VG and ZRP expression was a more sensitive reaction to the presence of NP than the formation of testis–ova and the reversal of sex. Increased VG expression in trout liver occurred already at 1.05 μg/l NP, whereas VG mRNA levels, quantified by competitive RT-PCR, were not significantly elevated in NP exposed fish. ZRP contents were significantly higher at 10.17 μg/l NP. Since induction of VG did not occur in all fish exposed to 1 or 10 μg/l NP and ZRP induction did not occur in all fish exposed to 10 μg/l, some individuals may be more affected by exposure to NP than others. This study demonstrates that NP concentrations typically found in sewage treatment effluents and some rivers do not affect sexual differentiation in rainbow trout, but induce VG and ZRP expression in the liver of exposed fish.     

Relationship between exposure duration,tissue residues,growth, and mortality in rainbow trout (Oncorhynchus mykiss) juveniles sub-chronically exposed to copper

We conducted a 56-day sub-chronic test on the effects of Cu on rainbow trout (Oncorhynchus mykiss) fry at a nominal water hardness of 100 mg l(-1) (as CaCO(3)). Response measures were growth, whole body Cu concentrations, and mortality. Significant mortality was observed in fish exposed to 54.1 microg Cu l(-1) (47.8%) and 35.7 microg Cu l(-1) (11.7%). Growth was dose-dependent over the range of Cu treatments (0-54 microg Cu l(-1)), and was modeled as a function of Cu exposure concentration and exposure duration. Calculated inhibition concentrations (based on change in wet weight through a 56-day Cu exposure) were IC(50)=54.0 microg Cu l(-1), IC(20)=21.6 microg Cu l(-1), IC(10)=10.8 microg Cu l(-1), and IC(01)=1.1 microg Cu l(-1). Measured whole body Cu was also dose-dependent, and growth of trout fry was readily modeled as a function of tissue Cu and exposure duration. This model was virtually identical to a model previously developed for rainbow trout exposed to Cu at a hardness of 25 mg l(-1). Following the 56-day exposure period, we performed a 96-h acute challenge to Cu and Cd to evaluate the effects of Cu acclimation on acute Cu and Cd toxicity. Sensitivity to Cu was dependent on the 'acclimation dose'; trout previously held in control aquaria (i.e. not acclimated to Cu) suffered over 80% mortality, whereas trout previously exposed to 35.7 microg Cu l(-1) for 56 day suffered 20% mortality. These fish also showed somewhat reduced sensitivity to Cd, suggesting acclimation to Cu can enhance tolerance to other metals. Finally, the relationship between growth response and hardness (derived from several studies) appeared to have a different slope than the hardness relationship previously observed for lethality responses.     

Changes in concentrations of microcystins in rainbow trout, freshwater mussels, and cyanobacteria in Lakes Rotoiti and Rotoehu

Microcystin concentrations in cyanobacteria and their accumulation in rainbow trout (Oncorhynchus mykiss) and freshwater mussels (Hyridella menziesi) in Lakes Rotoiti and Rotoehu (New Zealand) were investigated. Hatchery rainbow trout were added to an enclosure in Lake Rotoiti where concentrations of microcystins in the phytoplankton and cyanobacterial cell concentrations could be closely monitored. Rainbow trout that were free to roam in the entire area of each lake were also included in the study. Freshwater mussels were suspended subsurface in cages in the enclosure. Phytoplankton samples, rainbow trout liver and muscle tissue, and the tissues of mussels were analyzed for microcystins using the ADDA-ELISA method, and selected samples were analyzed using LC-MS. A maximum concentration of microcystins in the phytoplankton samples of 760 microg L(-1) was recorded in Te Weta Bay, Lake Rotoiti, in March 2004. ELISA results confirmed microcystin immunoreactivity in rainbow trout liver and muscle tissues and in freshwater mussels. The microcystin congeners LR, YR, RR, AR, FR, LA, and WR were detected by LC-MS in caged freshwater mussels in Lake Rotoiti but were not detected in either muscle or liver tissue of rainbow trout. The daily tolerable intake limit of microcystins for human consumption recommended by the World Health Organisation is 0.04 microg kg(-1) day(-1). Modeling was carried out for the human intake of microcystin compounds from rainbow trout muscle tissue, and the potential health risks were estimated, assuming the ADDA-ELISA was determining compounds of toxicity equivalent to microcystin-LR.     

Effect of nonylphenol on plasma vitellogenin of male adult guppies (Poecilia reticulata)

Adult male guppies (Poecilia reticulata) were exposed to 4-nonylphenol (NP) at nominal concentrations of 10, 60, and 150 microg/L for 7, 14, or 21 days. Significant induction of vitellogenin (VTG) was found in male guppies exposed to 1 microg/L of 17beta-estradiol and 60 or 150 microg/L of NP after 7, 14, or 21 days. Maximum induction was seen after 14 days of treatment at these concentrations. On the other hand, significant induction of VTG in male guppies exposed to 10 microg/L of NP was observed only after 21 days of treatment. A time-dependent tendency of reduction of the gonadosomatic index (GSI) in male guppies treated with 17beta-estradiol or NP was observed, but no corresponding concentration-dependent tendency was detected. There were also no differences in the liver somatic index (LSI) of male fish treated with various concentrations of NP during the above-mentioned exposure periods. Preliminary data presented in this study strongly suggests that measurement of plasma VTG induction in male guppies is a suitable and rather sensitive indicator of exposure to estrogenic chemicals.